Preparation method and application of urushiol oligomer

Abstract

The invention discloses a preparation method and application of a urushiol oligomer, and belongs to the technical field of oxidative polymerization of natural active matters. Raw lacquer is used as a raw material, the time and temperature of oxidative polymerization are controlled to obtain the urushiol polymer with oligomer as a main component, and then the urushiol oligomer with oxidation resistance and antibacterial activity is prepared through separation and purification. Wherein the polymerization degree of the urushiol oligomer is 2-5. By controlling the polymerization temperature and time of the raw lacquer and the urushiol, the polymerization degree of the urushiol is accurately controlled, and a crude extract mainly containing oligomers is formed; the urushiol oligomer with good antioxidant and antibacterial activity can be obtained through silica gel column chromatography, and the application of the urushiol oligomer in the fields of functional additives and the like is promoted, so that the application of raw lacquer and urushiol is expanded, and the bioavailability is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of oxidative polymerization of natural active substances, and more specifically, relates to a preparation method and application of urushiol oligomers. Background Art

[0002] Raw lacquer is the main secretion of lacquer trees. It has been used as a paint for thousands of years because of its good durability and glossiness. The curing of raw lacquer into film is a biochemical reaction process. Urushiol undergoes oxidative polymerization under the action of laccase. Catechol is first oxidized to form dimers, trimers and oligomers. When the concentration of lacquer phenol monomers is reduced to below 30%, the unsaturated side chains undergo auto-oxidation to form a bridging reaction, and then the enzymatic reaction and auto-oxidation are repeated to form a durable network polymer, thus forming a solid paint film.

[0003] Urushiol is the main component of raw lacquer, accounting for about 50% to 80% of the total raw lacquer. Urushiol has phenolic hydroxyl groups at C1 and C2, and a straight-chain alkane with 15-17 at C3. It is mainly composed of saturated urushiol and unsaturated urushiol (unsaturation ranges from 0 to 3). It is reported that urushiol has multiple effects such as anti-tumor, antibacterial, antiviral and antioxidant, and has great application potential in the field of functional additives. However, urushiol is very likely to cause allergic dermatitis. About 60% of the population is allergic to urushiol. After contact with raw lacquer, the skin will show symptoms such as swelling, erythema, and itching. In more severe cases, the skin will show burn-like blisters, which limits the utilization of urushiol.

[0004] Urushiol is a hapten, but when the hapten urushiol comes into contact with the skin, the catalytic reaction of the enzyme in the body oxidizes the urushiol to form an electrophilic o-quinone compound, which reacts with keratin or protein on the cell membrane at the 4, 5, and 6 positions of the benzene ring of urushiol to form a complete antigen. After the protein-urushiol complex forms a complete antigen, it is captured by Langerhans cells. The activated Langerhans cells gather in the lymph nodes and present the antigen information to effector T cells. These effector cells release cytokines, which bind to different target cells: macrophages, lymphocytes and other types of cells, and finally show pathological reactions such as contact dermatitis, erythema, papules, vesicles and swelling. The usual solution is to modify the phenolic hydroxyl group of urushiol to form esterification or acylation. Although it prevents the oxidation of urushiol to form o-quinone, its biological activity is significantly reduced. At the same time, chemical reactions will introduce some toxic and harmful substances, which is not conducive to application. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for preparing urushiol oligomers with antioxidant and antibacterial activities by using raw lacquer, which method has the advantages of simple operation, repeatable process and high oligomer yield. Another technical problem to be solved by the present invention is to provide the application of the above urushiol oligomers in the preparation of antioxidants and / or antibacterial agents.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for preparing urushiol oligomers, which uses raw lacquer as a raw material, controls the time and temperature of oxidative polymerization to obtain urushiol polymers mainly composed of oligomers, and then separates and purifies to prepare urushiol oligomers with antioxidant and antibacterial activities; wherein the polymerization degree of the urushiol oligomers is 2-5.

[0007] Preferably, the oxidative polymerization time is 20-60° C., and the oxidative polymerization time is 15 min-24 h.

[0008] Preferably, the oxidative polymerization time is 20-30° C., and the oxidative polymerization time is 8 h to 24 h.

[0009] Preferably, the oxidative polymerization time is 30-40° C., and the oxidative polymerization time is 4 h-6 h.

[0010] Preferably, the oxidative polymerization time is 40-50° C., and the oxidative polymerization time is 30 min-2 h.

[0011] Preferably, the oxidative polymerization time is 50-60° C., and the oxidative polymerization time is 15 min-1 h.

[0012] The method for preparing the urushiol oligomer specifically comprises the following steps: 1) placing the raw lacquer in a water bath for oxidative polymerization reaction, adding a solvent after the reaction is completed, mixing, centrifuging, and distilling the supernatant under reduced pressure to obtain a crude oligomer extract; 2) Dissolving the crude oligomer extract obtained in step 1) with petroleum ether, separating by silica gel column chromatography, eluting with a petroleum ether-ethyl acetate mixed solution, collecting the eluted portion, and distilling under reduced pressure to obtain urushiol oligomers.

[0013] Preferably, in step 1), the solvent is selected from one or more of ethanol, ethyl acetate, chloroform and tetrahydrofuran.

[0014] Preferably, in step 1), the mass volume ratio of raw lacquer to solvent is 1:10-20 g / mL.

[0015] Preferably, in step 1), the water bath is a temperature-controlled water bath with a magnetic stirring function, wherein the stirring speed is 200-600 rpm.

[0016] Preferably, in step 1), the temperature of the reduced pressure distillation is 30-50°C.

[0017] Preferably, in step 2), a petroleum ether-ethyl acetate mixed solution is used to elute 3 to 5 column volumes to remove impurities (unpolymerized urushiol monomers), wherein the volume ratio of petroleum ether to ethyl acetate is 9.8:0.2.

[0018] Preferably, in the step 2), the elution portion with a volume ratio of petroleum ether to ethyl acetate of 9.5:0.5 to 9.2:0.8 is collected to collect urushiol oligomers.

[0019] The method for preparing urushiol oligomers is used to prepare urushiol oligomers.

[0020] The urushiol oligomer is used in the preparation of antioxidants and / or antibacterial agents.

[0021] In order to reduce the allergenicity of urushiol and obtain substances with similar activity and application range to urushiol, the present invention adopts directed oxidative polymerization technology, and then obtains urushiol oligomers through separation, purification, preparation and other processes. Not only does it reduce the allergenicity of urushiol, but the urushiol oligomers also have good antioxidant activity and antibacterial activity.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1) The present invention controls the polymerization temperature and time of raw lacquer and urushiol, accurately controls the degree of urushiol polymerization, and forms a crude extract mainly composed of oligomers; then, urushiol oligomers with good antioxidant and antibacterial activities can be obtained by silica gel column chromatography, which is conducive to promoting its application in the fields of functional additives, thereby expanding the use of raw lacquer and urushiol and improving bioavailability; the method has the advantages of simple operation, repeatable process, and high oligomer yield; 2) The urushiol oligomer prepared by the present invention has antioxidant activity. When the concentration of the urushiol oligomer ethanol solution is in the range of 7.81-125 μg / mL, the scavenging rate of DPPH free radicals is 12.47-96.32%; when the concentration of the urushiol oligomer ethanol solution is in the range of 7.81-125 μg / mL, the scavenging rate of ABTS free radicals is 17.63-99.63%; 3) The degree of polymerization of the urushiol oligomer prepared by the present invention is 2-5, and the benzene ring of the urushiol is substituted to form a biphenyl type and a benzene ring-side chain coupling type, and its sensitization is greatly reduced, and it can be widely used in the fields of functional additives or materials with antioxidant and antibacterial properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is the result of scavenging DPPH free radicals by the urushiol oligomer prepared in Example 3; Figure 2 This is a graph showing the scavenging results of ABTS free radicals by the urushiol oligomer prepared in Example 3; Figure 3 These are the antibacterial effect diagrams of the urushiol oligomers prepared in Example 3; wherein, Figure A is the antibacterial effect diagram of the urushiol oligomers on Staphylococcus aureus; and Figure B is the antibacterial effect diagram of the urushiol oligomers on Staphylococcus epidermidis. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following examples, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0025] The raw lacquer adopted in the following examples is obtained from Toxicodendron suffruticosa (Latin name Toxicodendron vernicifluum , origin is Hubei) freshly collected.

[0026] The polymerization degree testing method adopted in the present invention is: using gel chromatography to perform polymerization degree analysis. Example 1

[0027] A method for preparing urushiol oligomers comprises the following steps: 1) Place the raw lacquer in a temperature-controlled water bath with magnetic stirring function, the temperature is controlled at 20-30 °C, the time is 8-24 h, and the stirring speed is 200-600 rpm; 2) Add ethanol at a mass volume ratio (g / mL) of 1:10 to the raw lacquer, stir evenly, centrifuge, and evaporate the supernatant under reduced pressure at 30°C to remove the solvent to obtain a crude oligomer extract; 3) Dissolve the crude oligomer extract obtained in step 2) with petroleum ether, then apply it to a silica gel column (3 cm×50 cm), elute with a volume ratio of petroleum ether:ethyl acetate of 9.8:0.2 for 3 times the column volume, collect the elution portion with a volume ratio of petroleum ether:ethyl acetate of 9.4:0.6, and evaporate under reduced pressure at 30°C to remove petroleum ether and ethyl acetate to obtain urushiol oligomers with a yield of 58.0% and a degree of polymerization of 2-5. Example 2

[0028] A method for preparing urushiol oligomers comprises the following steps: 1) Place the raw lacquer in a temperature-controlled water bath with magnetic stirring function, the temperature is controlled at 30-40°C, the time is 4-6 hours, and the stirring speed is 200-600 rpm; 2) Ethyl acetate was added at a mass volume ratio (g / mL) of 1:15 between the raw lacquer and ethyl acetate, and the mixture was stirred evenly, centrifuged, and the supernatant was evaporated under reduced pressure at 40°C to remove the solvent, thereby obtaining a crude oligomer extract; 3) Dissolve the crude oligomer extract obtained in step 2) with petroleum ether, then apply it to a silica gel column (3 cm×50 cm), elute with a petroleum ether:ethyl acetate volume ratio of 9.8:0.2 for 4 times the column volume, collect the elution portion with a petroleum ether:ethyl acetate volume ratio of 9.3:0.7, and evaporate under reduced pressure at 40°C to remove the petroleum ether and ethyl acetate to obtain urushiol oligomers with a yield of 52.0% and a degree of polymerization of 2-5. Example 3

[0029] A method for preparing urushiol oligomers comprises the following steps: 1) Place the raw lacquer in a temperature-controlled water bath with magnetic stirring function, the temperature is controlled at 40~50℃, the time is 30min~2h, and the stirring speed is 200~600 rpm; 2) Add chloroform at a mass volume ratio of raw lacquer to chloroform (g / mL) of 1:20, stir evenly, centrifuge, and evaporate the supernatant under reduced pressure at 50°C to remove the solvent to obtain a crude oligomer extract; 3) Dissolve the crude oligomer extract obtained in step 2) with petroleum ether, then apply it to a silica gel column (3 cm×50 cm), elute with a volume ratio of petroleum ether:ethyl acetate of 9.8:0.2 for 5 times the column volume, collect the elution portion with a volume ratio of petroleum ether:ethyl acetate of 9.5:0.5, and evaporate under reduced pressure at 50°C to remove petroleum ether and ethyl acetate to obtain urushiol oligomers with a yield of 55.0% and a degree of polymerization of 2-5. Example 4

[0030] A method for preparing urushiol oligomers comprises the following steps: 1) Place the raw lacquer in a temperature-controlled water bath with magnetic stirring function, the temperature is controlled at 50~60℃, the time is 15min~1h, and the stirring speed is 200~600 rpm; 2) Add tetrahydrofuran in a mass volume ratio (g / mL) of raw lacquer to tetrahydrofuran of 1:15, stir evenly, centrifuge, evaporate the supernatant under reduced pressure at 50°C to remove the solvent, and obtain a crude oligomer extract; 3) Dissolve the crude oligomer extract obtained in step 2) with petroleum ether, then apply it to a silica gel column (3 cm×50 cm), elute with a volume ratio of petroleum ether:ethyl acetate of 9.8:0.2 for 5 times the column volume, collect the elution portion with a volume ratio of petroleum ether:ethyl acetate of 9.2:0.8, evaporate under reduced pressure at 30°C to remove petroleum ether and ethyl acetate, and obtain urushiol oligomers with a yield of 51.0 and a degree of polymerization of 2-5. Example 5

[0031] A method for preparing urushiol oligomers comprises the following steps: 1) Place the raw lacquer in a temperature-controlled water bath with magnetic stirring function, the temperature is controlled at 20~60℃, the time is 15min~24h, and the stirring speed is 200~600 rpm; 2) Add ethanol at a mass volume ratio (g / mL) of 1:20 between raw lacquer and ethanol, stir evenly, centrifuge, and evaporate the supernatant under reduced pressure at 30°C to remove the solvent to obtain a crude oligomer extract; 3) Dissolve the crude oligomer extract obtained in step 2) with petroleum ether, then apply it to a silica gel column (3 cm×50 cm), elute with a volume ratio of petroleum ether:ethyl acetate of 9.8:0.2 for 3 times the column volume, collect the elution portion with a volume ratio of petroleum ether:ethyl acetate of 9.5:0.5, and evaporate under reduced pressure at 30°C to remove petroleum ether and ethyl acetate to obtain urushiol oligomers with a yield of 45.0% and a degree of polymerization of 2-5.

[0032] The urushiol oligomers prepared in Examples 1-5 were subjected to gel chromatography analysis, and the specific results obtained are shown in Table 1.

[0033] Table 1 Degree of polymerization of urushiol oligomers prepared in Examples 1-5 Example 6 1. DPPH free radical scavenging effect The antioxidant activity of urushiol oligomers was evaluated by scavenging DPPH free radicals. The test process was as follows: urushiol oligomers were dissolved in anhydrous ethanol and then diluted step by step to solutions with concentrations of 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 62.500 μg / mL, 31.250 μg / mL, 15.625 μg / mL, 7.813 μg / mL and 3.906 μg / mL. Vc and BHT were used as positive controls. 150 μL DPPH solution (0.15 mmol / L, dissolved in ethanol) was added to 50 μL of solutions of different concentrations, and the reaction was carried out at room temperature in the dark for 30 minutes, and the absorbance was measured at a wavelength of 517 nm. The calculation formula for the scavenging rate of DPPH free radicals is shown in formula (1): Formula (1); Where: T i T0 represents the absorbance of the sample and DPPH reaction solution; T1 represents the absorbance of DPPH in ethanol solvent. Figure 1 The results of Examples 1-5 are shown in Table 2.

[0034] Table 2 Results of scavenging DPPH free radicals From Table 2 and Figure 1 It can be seen that urushiol oligomers show a strong ability to scavenge DPPH free radicals, and their scavenging activity is positively correlated with the concentration of the oligomers. When the concentration of urushiol oligomer ethanol solution ranges from 7.81 to 125 μg / mL, the scavenging rate of DPPH free radicals is 12.47 to 96.32%, which is between Vc and BHT.

[0035] 2. ABTS free radical scavenging effect The antioxidant activity of urushiol oligomers was evaluated by scavenging ABTS free radicals. The test process was as follows: urushiol oligomers were dissolved in anhydrous ethanol and then diluted step by step to solutions with concentrations of 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 62.500 μg / mL, 31.250 μg / mL, 15.625 μg / mL, 7.813 μg / mL and 3.906 μg / mL. Vc and BHT were used as positive controls. 20 mL of ABTS (7.4 mmol / L, dissolved in ethanol) was mixed with 20 mL of potassium persulfate (2.6 mmol / L, dissolved in water) and reacted at room temperature in the dark for 15 h. The mixed solution was then diluted with ethanol to obtain a working solution (absorbance at 734 nm was 0.74 ± 0.02). 150 μL of the working solution was added to 50 μL of sample solutions of different concentrations, and the absorbance was measured at a wavelength of 734 nm. The calculation formula for ABTS free radical scavenging rate is shown in formula (2): Formula (2); Where: t i represents the absorbance of the sample and the ABTS working solution; t0 represents the absorbance of the sample in the ethanol solvent; t1 represents the absorbance of the ABTS working solution in the ethanol solvent. Figure 2 The results of Examples 1-5 are shown in Table 3.

[0036] Table 3 Results of scavenging ABTS free radicals From Table 3 and Figure 2It can be seen that urushiol oligomers show a strong ability to scavenge ABTS free radicals, and their scavenging activity is positively correlated with the concentration of the oligomers. When the concentration of urushiol oligomer ethanol solution ranges from 7.81 to 125 μg / mL, the scavenging rate of ABTS free radicals is 17.63% to 99.63%, which is between Vc and BHT.

[0037] 3. Antibacterial effect In the clean bench, Staphylococcus aureus and Staphylococcus epidermidis were inoculated into lysing broth (LB) liquid culture medium, and cultured in a shaker at 37°C at 120 r / min for 24 h. A trace amount of bacterial solution was taken and diluted with LB liquid culture medium. The concentration of the bacterial solution was measured with a turbidimeter and controlled at 10 8 CFU / mL, and then dilute 100 times for later use.

[0038] Urushiol oligomers were dissolved in 1% dimethyl sulfoxide aqueous solution and then diluted stepwise to solutions with concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL and 7.813 μg / mL. Take a 12×100 mm glass test tube, add 1 mL of urushiol oligomer solution of different concentrations to the test tube, and then add 1 mL of bacterial suspension to the test tube (the final bacterial solution concentration of each test tube is 5×10 5 CFU / mL). 1mL of 1% dimethyl sulfoxide aqueous solution and 1mL of bacterial suspension were used as blank controls. Gentamycin sulfate was used as a positive control. The test tubes were placed in a 37℃ constant temperature incubator for 24 hours, and then each test tube was observed. If the solution was clear, it indicated that the sample inhibited growth. The lowest concentration corresponding to the clear test tube was the minimum inhibitory concentration (MIC) of the sample for the bacteria.

[0039] The test tube was placed in a 37°C incubator for another 24 hours, and then 200 μL of the culture liquid of several concentrations near the clarified test tube was aspirated and cultured on LB solid medium. The culture was cultured in a 37°C incubator for 24 hours, and the bacterial growth was observed. The mass concentration corresponding to no colony growth was the minimum bactericidal concentration (MBC). The results of Example 3 are shown in FIG. Figure 3 The results of Examples 1-5 are shown in Table 4.

[0040] Depend on Figure 3 It can be seen that the minimum inhibitory concentration of the urushiol oligomer prepared in Example 3 against Staphylococcus aureus is 0.25 mg / mL, and the minimum bactericidal concentration is 1 mg / mL; the minimum inhibitory concentration against Staphylococcus epidermidis is 0.125 mg / mL, and the minimum bactericidal concentration is 0.5 mg / mL.

[0041] Table 4 Minimum inhibitory concentration and minimum bactericidal concentration for Staphylococcus aureus and Staphylococcus epidermidis The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing urushiol oligomers, characterized in that: Using raw lacquer as raw material, by controlling the time and temperature of oxidative polymerization, urushiol polymers mainly composed of oligomers are obtained, and then urushiol oligomers with antioxidant and antibacterial activities are prepared through separation and purification; wherein the degree of polymerization of urushiol oligomers is 2-5.

2. The method for preparing urushiol oligomers according to claim 1, characterized in that: The oxidative polymerization time is 20-60° C., and the oxidative polymerization time is 15 min-24 h.

3. The method for preparing urushiol oligomers according to claim 2, characterized in that: The oxidative polymerization time is 20-30° C., and the oxidative polymerization time is 8 h to 24 h.

4. The method for preparing urushiol oligomers according to claim 2, characterized in that: The oxidative polymerization time is 30-40° C., and the oxidative polymerization time is 4h-6h.

5. The method for preparing urushiol oligomers according to claim 2, characterized in that: The oxidative polymerization time is 40-50° C., and the oxidative polymerization time is 30 min-2 h.

6. The method for preparing urushiol oligomers according to claim 2, characterized in that: The oxidative polymerization time is 50-60° C., and the oxidative polymerization time is 15 min-1 h.

7. The method for preparing urushiol oligomers according to claim 1, characterized in that: The specific steps include: 1) placing the raw lacquer in a water bath for oxidative polymerization reaction, adding a solvent after the reaction is completed, mixing, centrifuging, and distilling the supernatant under reduced pressure to obtain a crude oligomer extract; 2) Dissolving the crude oligomer extract obtained in step 1) with petroleum ether, separating by silica gel column chromatography, eluting with a petroleum ether-ethyl acetate mixed solution, collecting the eluted portion, and distilling under reduced pressure to obtain urushiol oligomers.

8. The method for preparing urushiol oligomers according to claim 7, characterized in that: In the step 1), the solvent is selected from one or more of ethanol, ethyl acetate, chloroform and tetrahydrofuran.

9. The method for preparing the urushiol oligomer according to any one of claims 1 to 8, wherein the urushiol oligomer is prepared.

10. Use of the urushiol oligomer according to claim 9 in the preparation of an antioxidant and / or an antibacterial agent.

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